PROJECT SUMMARY/ABSTRACT Here we seek to investigate components directly involved in activation of Stimulator of Interferon Genes (STING), an endoplasmic reticulum trans-membrane protein that is responsible for key events in immunopathogenesis of early alcoholic liver disease. Activation of STING leads to phosphorylation of Interferon Regulatory Factor 3 (IRF3), a transcription factor that plays an important role in the activation of innate immunity and in activation pro-apoptotic signaling in hepatocytes after ethanol administration. In immune cells, STING is activated by cyclic dinucleotides. 2'3'-cGAMP is a mammalian cyclic dinucleotide synthesized by cyclic GMP-AMP synthase (cGAS), an intracellular dsDNA sensor. c-di-GMP, the bacterial dinucleotide, is synthesized by diguanylate cyclase (DGC) in over 90% of bacteria. Recently, cyclic dinucleotides have been shown to pass from cell to cell through gap junctions. We have previously reported that mice deficient in STING or IRF3 are protected from alcoholic liver disease. Here, we show that mice deficient in cGAS are protected from acute or acute-on-chronic ethanol-induced liver injury and phosphorylation of IRF3. We also show that pharmacological inhibition of hepatic gap junctions protects from acute or acute-on-chronic ethanol-induced liver injury. We show evidence that both mammalian and bacterial cyclic dinucleotides are capable of activation STING and IRF3 in primary hepatocytes. Lastly, we show that gap junctions propagate ethanol-induced phosphorylation of IRF3 to neighboring cells in vitro. Based on these findings, we hypothesize that cyclic dinucleotides from the host animal and gut microbiome converge in activation of STING and IRF3 in early alcoholic liver disease. The aims proposed as part of this research proposal encompass basic molecular biology, analytical chemistry, as well as the use of small-molecule inhibitors and viral vector-mediated gene transfer technology as therapeutic interventions in two models of early alcoholic liver disease in mice. The main objectives can be summarized as follows: 1. Detect and quantify mammalian and bacterial cyclic dinucleotides in serum, whole liver lysate after ethanol-induced liver injury. 2. Study the spatiotemporal dynamics of gap junction-mediated cyclic dinucleotides that lead to ethanol- induced STING and IRF3 activation in the liver. 3. Characterize the potential of bacterial diguanylate cyclase and its associated cyclic dinucleotides as viable therapeutic targets in the treatment of early alcoholic liver disease using small-molecule inhibitors and/or adeno-associated virus associated gene therapy.